DESCRIPTION: The MyoD gene family encodes muscle-specific transcription factors that are essential for skeletal muscle development and regeneration. MyoD or the related gene, Myf5, is required to establish all skeletal muscle lineages in mouse embryos. In addition, MyoD is indispensable for the regeneration of injured and dystrophic muscle. The long-term objective of the proposed research is to elucidate the molecular mechanisms that regulate the transcription of the MyoD gene during muscle development and regeneration. Two transcriptional enhancers [the core enhancer at -20 kb and distal regulatory region (DRR) at -5 kb] have been implicated in the control of MyoD gene expression, although the functional importance of these enhancers within their normal chromosomal context has not been investigated. In this proposal, homologous recombination in embryonic stem cells will be used to produce mice with a targeted deletion of one or both enhancers. Essential enhancer functions will be elucidated by analyzing MyoD expression, muscle formation, and regeneration in these mutant mice. MyoD expression in developing muscle will be examined by quantitative Northern and RNase protection assays to distinguish whether unique, cooperative, or redundant enhancer functions control the level and timing of MyoD expression. In situ hybridization will determine whether expression in distinct muscle lineages is controlled by differential utilization of the core enhancer and DRR. The consequences of enhancer deletions on muscle formation will be analyzed by immunohistochemistry in a Myf5 null background, where myogenesis is completely dependent on MyoD function. The consequences of enhancer deletions on MyoD expression in injured muscle and in dystrophic muscle of mdx mice (mouse model for Duchenne muscular dystrophy) will also be determined. Concurrently, muscle regeneration will be evaluated by histological and morphometric criteria, as a sensitive bioassay for enhancer function. The proposed studies will determine how the MyoD gene is transcriptionally regulated, providing a framework by which to understand abnormal developmental processes and regenerative responses to muscle injury and disease.